SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017 - 1208

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SLAS Discovery 22(10)

Figure 3. Comparison of in vitro c-MET kinase assay analyzed by MALDI-MS or Caliper LabChip 3000 CE. c-MET assay carried out
in MALDI-MS-compatible buffer conditions. IC50 values were determined for selected compounds by testing SRCtide phosphorylation
in the presence of a range of inhibitor concentrations. (A,B) Representative fitted dose-response curves for three compounds
analyzed by (A) MALDI-MS and (B) Caliper LabChip 3000 CE. (C) Overview of IC50 values for all tested compounds and (D)
correlation of obtained IC50 values by either MALDI-MS or Caliper CE.

(pH 7.5), 1 mM MgCl2, 2 mM DTT, and 0.01% Brij-35 for
our c-MET kinase assay. This resulted in final concentrations in the sample-matrix mix of 0.5 mM HEPES (pH 7.5),
0.5 mM MgCl2, 1 mM DTT, and 0.005% Brij-35.
The rationale for the use of MALDI-MS in hit discovery
is a fast, unbiased, and reliable detection of assay components. Enzyme assays that modify a substrate to produce a
product with a clear mass shift are well suited for
MALDI-MS. For the c-MET kinase assay described here,
we used SRCtide, a well-established tyrosine kinase peptide, as a substrate. The c-MET kinase phosphorylates the
SRCtide peptide at the tyrosine, resulting in p-SRCtide
(Fig. 2A). Corresponding to this enzymatic activity, a mass
increase of 80 Da for an added phosphate was detected and
produces a mass shift of m/z = 80 in the mass profile.
Consequently, both SRC and p-SRC can be detected individually/independently but in parallel via MALDI-MS
(Fig. 2B). Even though the appearance of the mass shift
indicates enzyme activity, quantitative measurement of the
substrate (SRC) and product (p-SRC) is needed for our
screening purposes in drug discovery. Additionally, to
allow evaluation of enzyme activities between sample
measurements, peak intensities have to be comparable
from one well to another. It has been shown that MALDI
efficiencies for phosphopeptides are significantly different
than those of the nonphosphorylated forms.17,18 To assess

the influence of desorption/ionization efficiencies for the
c-MET assay, we used our established setup and compared
the detection of SRC and p-SRC over a range of concentrations (Fig. 2C,D). As expected, the raw data show a difference in signal intensity between the nonphosphorylated
and phosphorylated peptides. However, the signal intensity
ratio is mostly consistent over the tested concentration
range. Therefore, we expect that normalization achieves
comparability between wells (Fig. 2D). Normalization can
be done either by addition of an internal marker to each
reaction or by normalization to controls on the plate.
Furthermore, within the linear range shown in Figure 2D a
quantitative relationship between the intensity of SRC or
p-SRC as measured by MALDI-MS and the assay concentration exists. Consequently, the ratio of peak intensities
measured for the substrate and product represents a linear
relationship to enzyme activity within the assay. Hence,
our experimental results were consistent with predictions
allowing direct assessment and comparison of c-MET
enzyme activities, which is especially useful in HTS
assays.
Another advantage of using MALDI-MS for hit discovery is that it enables screening compounds for their influence on enzymatic activity in a direct, fast, and reliable way.
After we determined suitable assay conditions (Fig. 1) and
a linear detection range for substrate and product (Fig. 2),

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